RESUMEN
Macropinocytosis is defined as an actin-dependent but coat- and dynamin-independent endocytic uptake process, which generates large intracellular vesicles (macropinosomes) containing a non-selective sampling of extracellular fluid. Macropinocytosis provides an important mechanism of immune surveillance by dendritic cells and macrophages, but also serves as an essential nutrient uptake pathway for unicellular organisms and tumor cells. This review examines the cell biological mechanisms that drive macropinocytosis, as well as the complex signaling pathways - GTPases, lipid and protein kinases and phosphatases, and actin regulatory proteins - that regulate macropinosome formation, internalization, and disposition.
Asunto(s)
Actinas , Pinocitosis , Endocitosis , Transducción de Señal , MacrófagosRESUMEN
PI3Kß is required for invadopodia-mediated matrix degradation by breast cancer cells. Invadopodia maturation requires GPCR activation of PI3Kß and its coupling to SHIP2 to produce PI(3,4)P2 . We now test whether selectivity for PI3Kß is preserved under conditions of mutational increases in PI3K activity. In breast cancer cells where PI3Kß is inhibited, short-chain diC8-PIP3 rescues gelatin degradation in a SHIP2-dependent manner; rescue by diC8-PI(3,4)P2 is SHIP2-independent. Surprisingly, the expression of either activated PI3Kß or PI3Kα mutants rescued the effects of PI3Kß inhibition. In both cases, gelatin degradation was SHIP2-dependent. These data confirm the requirement for PIP3 conversion to PI(3,4)P2 for invadopodia function and suggest that selectivity for distinct PI3K isotypes may be obviated by mutational activation of the PI3K pathway.
Asunto(s)
Fosfatidilinositol 3-Quinasa Clase I/genética , Matriz Extracelular/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas/genética , Podosomas/metabolismo , Línea Celular Tumoral , Movimiento Celular , Fosfatidilinositol 3-Quinasa Clase I/metabolismo , Diglicéridos/química , Matriz Extracelular/ultraestructura , Femenino , Regulación de la Expresión Génica , Células HEK293 , Humanos , Glándulas Mamarias Humanas/citología , Glándulas Mamarias Humanas/metabolismo , Mutación , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas/metabolismo , Podosomas/ultraestructura , Transducción de SeñalRESUMEN
S100A4, a member of the S100 family of multifunctional calcium-binding proteins, participates in several physiological and pathological processes. In this study, we demonstrate that S100A4 expression is robustly induced in differentiating fiber cells of the ocular lens and that S100A4 (-/-) knockout mice develop late-onset cortical cataracts. Transcriptome profiling of lenses from S100A4 (-/-) mice revealed a robust increase in the expression of multiple photoreceptor- and Müller glia-specific genes, as well as the olfactory sensory neuron-specific gene, S100A5. This aberrant transcriptional profile is characterized by corresponding increases in the levels of proteins encoded by the aberrantly upregulated genes. Ingenuity pathway network and curated pathway analyses of differentially expressed genes in S100A4 (-/-) lenses identified Crx and Nrl transcription factors as the most significant upstream regulators, and revealed that many of the upregulated genes possess promoters containing a high-density of CpG islands bearing trimethylation marks at histone H3K27 and/or H3K4, respectively. In support of this finding, we further documented that S100A4 (-/-) knockout lenses have altered levels of trimethylated H3K27 and H3K4. Taken together, our findings suggest that S100A4 suppresses the expression of retinal genes during lens differentiation plausibly via a mechanism involving changes in histone methylation.
Asunto(s)
Catarata/patología , Diferenciación Celular , Cristalino/metabolismo , Retina/patología , Proteína de Unión al Calcio S100A4/deficiencia , Citoesqueleto de Actina/metabolismo , Animales , Transporte Biológico , Calcio/metabolismo , Catarata/genética , Linaje de la Célula/genética , Células Ependimogliales/metabolismo , Uniones Comunicantes/metabolismo , Eliminación de Gen , Ácido Glutámico/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Metilación , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Biológicos , Neuronas Receptoras Olfatorias/metabolismo , Especificidad de Órganos , Células Fotorreceptoras de Vertebrados/metabolismo , Análisis de Componente Principal , Proteína de Unión al Calcio S100A4/genética , Proteína de Unión al Calcio S100A4/metabolismo , Transcriptoma/genética , Regulación hacia Arriba/genéticaRESUMEN
Macropinocytosis is an actin-dependent but clathrin-independent endocytic process by which cells nonselectively take up large aliquots of extracellular material. Macropinocytosis is used for immune surveillance by dendritic cells, as a route of infection by viruses and protozoa, and as a nutrient uptake pathway in tumor cells. In this study, we explore the role of class I phosphoinositide 3-kinases (PI3Ks) during ligand-stimulated macropinocytosis. We find that macropinocytosis in response to receptor tyrosine kinase activation is strikingly dependent on a single class I PI3K isoform, namely PI3Kß (containing the p110ß catalytic subunit encoded by PIK3CB). Loss of PI3Kß expression or activity blocks macropinocytosis at early steps, before the formation of circular dorsal ruffles, but also plays a role in later steps, downstream from Rac1 activation. PI3Kß is also required for the elevated levels of constitutive macropinocytosis found in tumor cells that are defective for the PTEN tumor suppressor. Our data shed new light on PI3K signaling during macropinocytosis, and suggest new therapeutic uses for pharmacological inhibitors of PI3Kß.
Asunto(s)
Fosfatidilinositol 3-Quinasa Clase I/biosíntesis , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Pinocitosis , Transducción de Señal , Animales , Fosfatidilinositol 3-Quinasa Clase I/genética , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Ratones , Células 3T3 NIH , Neuropéptidos/genética , Neuropéptidos/metabolismo , Células PC-3 , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rac1/metabolismoRESUMEN
The invasion of tumor cells from the primary tumor is mediated by invadopodia, actin-rich protrusive organelles that secrete matrix metalloproteases and degrade the extracellular matrix. This coupling between protrusive activity and matrix degradation facilitates tumor invasion. We previously reported that the PI3Kß isoform of PI 3-kinase, which is regulated by both receptor tyrosine kinases and G protein-coupled receptors, is required for invasion and gelatin degradation in breast cancer cells. We have now defined the mechanism by which PI3Kß regulates invadopodia. We find that PI3Kß is specifically activated downstream from integrins, and is required for integrin-stimulated spreading and haptotaxis as well as integrin-stimulated invadopodia formation. Surprisingly, these integrin-stimulated and PI3Kß-dependent responses require the production of PI(3,4)P2 by the phosphoinositide 5'-phosphatase SHIP2. Thus, integrin activation of PI3Kß is coupled to the SHIP2-dependent production of PI(3,4)P2, which regulates the recruitment of PH domain-containing scaffolds such as lamellipodin to invadopodia. These findings provide novel mechanistic insight into the role of PI3Kß in the regulation of invadopodia in breast cancer cells.
Asunto(s)
Fosfatidilinositol 3-Quinasa/metabolismo , Fosfatidilinositoles/metabolismo , Podosomas/metabolismo , Actinas/metabolismo , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Quimiotaxis/fisiología , Matriz Extracelular/metabolismo , Femenino , Humanos , Integrinas/metabolismo , Integrinas/fisiología , Invasividad Neoplásica/patología , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Podosomas/fisiología , Transducción de Señal/fisiologíaRESUMEN
The phosphoinositide 3-kinase (PI3K) family includes eight distinct catalytic subunits and seven regulatory subunits. Only two PI3Ks are directly regulated downstream from G protein-coupled receptors (GPCRs): the class I enzymes PI3Kß and PI3Kγ. Both enzymes produce phosphatidylinositol 3,4,5-trisposphate in vivo and are regulated by both heterotrimeric G proteins and small GTPases from the Ras or Rho families. However, PI3Kß is also regulated by direct interactions with receptor tyrosine kinases (RTKs) and their tyrosine phosphorylated substrates, and similar to the class II and III PI3Ks, it binds activated Rab5. The unusually complex regulation of PI3Kß by small and trimeric G proteins and RTKs leads to a rich landscape of signaling responses at the cellular and organismic levels. This review focuses first on the regulation of PI3Kß activity in vitro and in cells, and then summarizes the biology of PI3Kß signaling in distinct tissues and in human disease.
Asunto(s)
Fosfatidilinositol 3-Quinasa Clase Ia/metabolismo , Animales , Fosfatidilinositol 3-Quinasa Clase Ia/química , Fosfatidilinositol 3-Quinasa Clase Ia/genética , Activación Enzimática , GTP Fosfohidrolasas/metabolismo , Humanos , Estructura Molecular , Mutación , Neoplasias/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transducción de SeñalRESUMEN
Phosphoinositide 3-kinase ß (PI3Kß) is regulated by receptor tyrosine kinases (RTKs), G protein-coupled receptors (GPCRs), and small GTPases such as Rac1 and Rab5. Our lab previously identified two residues (Gln596 and Ile597) in the helical domain of the catalytic subunit (p110ß) of PI3Kß whose mutation disrupts binding to Rab5. To better define the Rab5-p110ß interface, we performed alanine-scanning mutagenesis and analyzed Rab5 binding with an in vitro pulldown assay with GST-Rab5GTP Of the 35 p110ß helical domain mutants assayed, 11 disrupted binding to Rab5 without affecting Rac1 binding, basal lipid kinase activity, or Gßγ-stimulated kinase activity. These mutants defined the Rab5-binding interface within p110ß as consisting of two perpendicular α-helices in the helical domain that are adjacent to the initially identified Gln596 and Ile597 residues. Analysis of the Rab5-PI3Kß interaction by hydrogen-deuterium exchange MS identified p110ß peptides that overlap with these helices; no interactions were detected between Rab5 and other regions of p110ß or p85α. Similarly, the binding of Rab5 to isolated p85α could not be detected, and mutations in the Ras-binding domain (RBD) of p110ß had no effect on Rab5 binding. Whereas soluble Rab5 did not affect PI3Kß activity in vitro, the interaction of these two proteins was critical for chemotaxis, invasion, and gelatin degradation by breast cancer cells. Our results define a single, discrete Rab5-binding site in the p110ß helical domain, which may be useful for generating inhibitors to better define the physiological role of Rab5-PI3Kß coupling in vivo.
Asunto(s)
Neoplasias de la Mama/patología , Invasividad Neoplásica , Fosfatidilinositol 3-Quinasa/metabolismo , Proteínas de Unión al GTP rab5/metabolismo , Sitios de Unión , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Quimiotaxis , Gelatina/metabolismo , Células HEK293 , Humanos , Espectrometría de Masas/métodos , Mutación , Fosfatidilinositol 3-Quinasa/genética , Unión ProteicaRESUMEN
Nonmuscle myosin-IIA (NMHC-IIA) heavy chain phosphorylation has gained recognition as an important feature of myosin-II regulation. In previous work, we showed that phosphorylation on S1943 promotes myosin-IIA filament disassembly in vitro and enhances EGF-stimulated lamellipod extension of breast tumor cells. However, the contribution of NMHC-IIA S1943 phosphorylation to the modulation of invasive cellular behavior and metastasis has not been examined. Stable expression of phosphomimetic (S1943E) or non-phosphorylatable (S1943A) NMHC-IIA in breast cancer cells revealed that S1943 phosphorylation enhances invadopodia function, and is critical for matrix degradation in vitro and experimental metastasis in vivo. These studies demonstrate a novel link between NMHC-IIA S1943 phosphorylation, the regulation of extracellular matrix degradation and tumor cell invasion and metastasis.
Asunto(s)
Proteínas del Citoesqueleto/metabolismo , Metástasis de la Neoplasia/patología , Miosina Tipo IIA no Muscular/metabolismo , Podosomas/metabolismo , Línea Celular Tumoral , Proliferación Celular/fisiología , Humanos , Cadenas Pesadas de Miosina/metabolismo , Fosforilación , Podosomas/genéticaRESUMEN
S100A4, a member of the S100 family of Ca2+-binding proteins, is a key regulator of cell migration and invasion. Our previous studies showed that bone marrow-derived macrophages from S100A4-/- mice exhibit defects in directional motility and chemotaxis in vitro and reduced recruitment to sites of inflammation in vivo. We now show that the loss of S100A4 produces two mechanistically distinct phenotypes with regard to macrophage invasion: a defect in matrix degradation, due to a disruption of podosome rosettes caused by myosin-IIA overassembly, and a myosin-independent increase in microtubule acetylation, which increases podosome rosette stability and is sufficient to inhibit macrophage invasion. Our studies point to S100A4 as a critical regulator of matrix degradation, whose actions converge on the dynamics and degradative functions of podosome rosettes.
Asunto(s)
Movimiento Celular , Macrófagos/metabolismo , Miosina Tipo IIA no Muscular/metabolismo , Proteína de Unión al Calcio S100A4/metabolismo , Animales , Quimiotaxis , Ratones , Modelos Moleculares , Multimerización de Proteína , Proteína de Unión al Calcio S100A4/genéticaRESUMEN
Vps34 PI3K is thought to be the main producer of phosphatidylinositol-3-monophosphate, a lipid that controls intracellular vesicular trafficking. The organismal impact of systemic inhibition of Vps34 kinase activity is not completely understood. Here we show that heterozygous Vps34 kinase-dead mice are healthy and display a robustly enhanced insulin sensitivity and glucose tolerance, phenotypes mimicked by a selective Vps34 inhibitor in wild-type mice. The underlying mechanism of insulin sensitization is multifactorial and not through the canonical insulin/Akt pathway. Vps34 inhibition alters cellular energy metabolism, activating the AMPK pathway in liver and muscle. In liver, Vps34 inactivation mildly dampens autophagy, limiting substrate availability for mitochondrial respiration and reducing gluconeogenesis. In muscle, Vps34 inactivation triggers a metabolic switch from oxidative phosphorylation towards glycolysis and enhanced glucose uptake. Our study identifies Vps34 as a new drug target for insulin resistance in Type-2 diabetes, in which the unmet therapeutic need remains substantial.
Asunto(s)
Resistencia a la Insulina , Mitocondrias/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Transducción de Señal/fisiología , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Autofagia/fisiología , Línea Celular Tumoral , Fosfatidilinositol 3-Quinasas Clase III , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Técnicas de Sustitución del Gen , Glucosa/análisis , Glucosa/metabolismo , Prueba de Tolerancia a la Glucosa , Glucólisis/fisiología , Hepatocitos , Heterocigoto , Humanos , Insulina/metabolismo , Hígado/citología , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Modelos Animales , Músculo Esquelético/citología , Músculo Esquelético/metabolismo , Mioblastos , Fosfatidilinositol 3-Quinasas/genética , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación , Cultivo Primario de CélulasRESUMEN
Phosphoinositide 3-kinases (PI 3-kinases) are regulated by a diverse range of upstream activators, including receptor tyrosine kinases (RTKs), G-protein-coupled receptors (GPCRs), and small GTPases from the Ras, Rho and Rab families. For the Class IA PI 3-kinase PI3Kß, two mechanisms for GPCR-mediated regulation have been described: direct binding of Gßγ subunits to the C2-helical domain linker of p110ß, and Dock180/Elmo1-mediated activation of Rac1, which binds to the Ras-Binding Domain of p110ß. We now show that the integration of these dual pathways is unexpectedly complex. In breast cancer cells, expression of constitutively activated Rac1 (CA-Rac1) along with either GPCR stimulation or expression of Gßγ led to an additive PI3Kß-dependent activation of Akt. Whereas CA-Rac1-mediated activation of Akt was blocked in cells expressing a mutated PI3Kß that cannot bind Gßγ, Gßγ and GPCR-mediated activation of Akt was preserved when Rac1 binding to PI3Kß was blocked. Surprisingly, PI3Kß-dependent CA-Rac1 signaling to Akt was still seen in cells expressing a mutant p110ß that cannot bind Rac1. Instead of directly binding to PI3Kß, CA-Rac1 acts by enhancing Gßγ coupling to PI3Kß, as CA-Rac1-mediated Akt activation was blocked by inhibitors of Gßγ. Cells expressing CA-Rac1 exhibited a robust induction of macropinocytosis, and inhibitors of macropinocytosis blocked the activation of Akt by CA-Rac1 or lysophosphatidic acid. Our data suggest that Rac1 can potentiate the activation of PI3Kß by GPCRs through an indirect mechanism, by driving the formation of macropinosomes that serve as signaling platforms for Gßγ coupling to PI3Kß.
Asunto(s)
Fosfatidilinositol 3-Quinasa Clase Ia/metabolismo , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Pinocitosis/fisiología , Transducción de Señal/fisiología , Proteína de Unión al GTP rac1/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Línea Celular Tumoral , Fosfatidilinositol 3-Quinasa Clase Ia/genética , Activación Enzimática/genética , Subunidades beta de la Proteína de Unión al GTP/genética , Subunidades gamma de la Proteína de Unión al GTP/genética , Células HEK293 , Humanos , Lisofosfolípidos/genética , Lisofosfolípidos/metabolismo , Unión Proteica , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas de Unión al GTP rac/genética , Proteínas de Unión al GTP rac/metabolismo , Proteína de Unión al GTP rac1/genéticaRESUMEN
Class I phosphoinositide 3-kinases (PI3Ks) catalyze production of the lipid messenger phosphatidylinositol 3,4,5-trisphosphate (PIP3), which plays a central role in a complex signaling network regulating cell growth, survival, and movement. This network is overactivated in cancer and inflammation, and there is interest in determining the PI3K catalytic subunit (p110α, p110ß, p110γ, or p110δ) that should be targeted in different therapeutic contexts. Previous studies have defined unique regulatory inputs for p110ß, including direct interaction with Gßγ subunits, Rac, and Rab5. We generated mice with knock-in mutations of p110ß that selectively blocked the interaction with Gßγ and investigated its contribution to the PI3K isoform dependency of receptor tyrosine kinase (RTK) and G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor (GPCR) responses in primary macrophages and neutrophils. We discovered a unique role for p110ß in supporting synergistic PIP3 formation in response to the coactivation of macrophages by macrophage colony-stimulating factor (M-CSF) and the complement protein C5a. In contrast, we found partially redundant roles for p110α, p110ß, and p110δ downstream of M-CSF alone and a nonredundant role for p110γ downstream of C5a alone. This role for p110ß completely depended on direct interaction with Gßγ, suggesting that p110ß transduces GPCR signals in the context of coincident activation by an RTK. The p110ß-Gßγ interaction was also required for neutrophils to generate reactive oxygen species in response to the Fcγ receptor-dependent recognition of immune complexes and for their ß2 integrin-mediated adhesion to fibrinogen or poly-RGD+, directly implicating heterotrimeric G proteins in these two responses.
Asunto(s)
Células Mieloides/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal , Animales , Ratones , Ratones Noqueados , Fosfatidilinositol 3-Quinasas/genética , Receptores Acoplados a Proteínas G/genética , Proteínas de Unión al GTP rab5/genética , Proteínas de Unión al GTP rab5/metabolismo , Proteínas de Unión al GTP rac/genética , Proteínas de Unión al GTP rac/metabolismoRESUMEN
The Class III phosphoinositide 3-kinase Vps34 (vacuolar protein sorting 34) plays important roles in endocytic trafficking, macroautophagy, phagocytosis, cytokinesis and nutrient sensing. Recent studies have provided exciting new insights into the structure and regulation of this lipid kinase, and new cellular functions for Vps34 have emerged. This review critically examines the wealth of new data on this important enzyme, and attempts to integrate these findings with current models of Vps34 signalling.
Asunto(s)
Fosfatidilinositol 3-Quinasas Clase III/metabolismo , Animales , Autofagia , Fosfatidilinositol 3-Quinasas Clase III/química , Endosomas/enzimología , Humanos , Fosforilación , Conformación ProteicaRESUMEN
Inappropriate activation of PI3K signaling has been implicated strongly in human cancer. Although studies on the role of PI3K signaling in breast tumorigenesis and progression have focused most intensively on PI3Kα, a role for PI3Kß has begun to emerge. The PI3Kß isoform is unique among class IA PI3K enzymes in that it is activated by both receptor tyrosine kinases and G-protein-coupled receptors (GPCR). In previous work, we identified a mutation that specifically abolishes PI3Kß binding to Gßγ (p110(526KK-DD)). Expression of this mutant in p110ß-silenced breast cancer cells inhibits multiple steps of the metastatic cascade in vitro and in vivo and causes a cell autonomous defect in invadopodial matrix degradation. Our results identify a novel link between GPCRs and PI3Kß in mediating metastasis, suggesting that disruption of this link might offer a novel therapeutic target to prevent the development of metastatic disease. Cancer Res; 76(10); 2944-53. ©2016 AACR.
Asunto(s)
Neoplasias de la Mama/patología , Transformación Celular Neoplásica/patología , Fosfatidilinositol 3-Quinasa Clase Ia/metabolismo , Neoplasias Pulmonares/secundario , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal , Animales , Apoptosis , Western Blotting , Neoplasias de la Mama/metabolismo , Movimiento Celular , Proliferación Celular , Transformación Celular Neoplásica/metabolismo , Femenino , Humanos , Neoplasias Pulmonares/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones SCID , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
In contrast to the class I phosphoinositide 3-kinases (PI3Ks), the organismal roles of the kinase activity of the class II PI3Ks are less clear. Here, we report that class II PI3K-C2ß kinase-dead mice are viable and healthy but display an unanticipated enhanced insulin sensitivity and glucose tolerance, as well as protection against high-fat-diet-induced liver steatosis. Despite having a broad tissue distribution, systemic PI3K-C2ß inhibition selectively enhances insulin signaling only in metabolic tissues. In a primary hepatocyte model, basal PI3P lipid levels are reduced by 60% upon PI3K-C2ß inhibition. This results in an expansion of the very early APPL1-positive endosomal compartment and altered insulin receptor trafficking, correlating with an amplification of insulin-induced, class I PI3K-dependent Akt signaling, without impacting MAPK activity. These data reveal PI3K-C2ß as a critical regulator of endosomal trafficking, specifically in insulin signaling, and identify PI3K-C2ß as a potential drug target for insulin sensitization.
Asunto(s)
Fosfatidilinositol 3-Quinasas Clase II/metabolismo , Insulina/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Autofagia , Glucemia/análisis , Células Cultivadas , Fosfatidilinositol 3-Quinasas Clase II/genética , Dieta Alta en Grasa , Endosomas/metabolismo , Hígado Graso/metabolismo , Hígado Graso/patología , Técnicas de Sustitución del Gen , Hepatocitos/citología , Hepatocitos/metabolismo , Insulina/sangre , Hígado/patología , Ratones , Ratones Endogámicos C57BL , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de SeñalRESUMEN
Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that are activated by growth factor and G-protein-coupled receptors and propagate intracellular signals for growth, survival, proliferation, and metabolism. p85α, a modular protein consisting of five domains, binds and inhibits the enzymatic activity of class IA PI3K catalytic subunits. Here, we describe the structural states of the p85α dimer, based on data from in vivo and in vitro solution characterization. Our in vitro assembly and structural analyses have been enabled by the creation of cysteine-free p85α that is functionally equivalent to native p85α. Analytical ultracentrifugation studies showed that p85α undergoes rapidly reversible monomer-dimer assembly that is highly exothermic in nature. In addition to the documented SH3-PR1 dimerization interaction, we identified a second intermolecular interaction mediated by cSH2 domains at the C-terminal end of the polypeptide. We have demonstrated in vivo concentration-dependent dimerization of p85α using fluorescence fluctuation spectroscopy. Finally, we have defined solution conditions under which the protein is predominantly monomeric or dimeric, providing the basis for small angle x-ray scattering and chemical cross-linking structural analysis of the discrete dimer. These experimental data have been used for the integrative structure determination of the p85α dimer. Our study provides new insight into the structure and assembly of the p85α homodimer and suggests that this protein is a highly dynamic molecule whose conformational flexibility allows it to transiently associate with multiple binding proteins.
Asunto(s)
Fosfatidilinositol 3-Quinasa Clase Ia/química , Multimerización de Proteína , Fosfatidilinositol 3-Quinasa Clase Ia/genética , Fosfatidilinositol 3-Quinasa Clase Ia/metabolismo , Humanos , Estructura Cuaternaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
In the liver, insulin-mediated activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway is at the core of metabolic control. Multiple PI3K and Akt isoenzymes are found in hepatocytes and whether isoform-selective interplays exist is currently unclear. Here we report that insulin signalling triggers the association of the liver-specific class II PI3K isoform γ (PI3K-C2γ) with Rab5-GTP, and its recruitment to Rab5-positive early endosomes. In these vesicles, PI3K-C2γ produces a phosphatidylinositol-3,4-bisphosphate pool specifically required for delayed and sustained endosomal Akt2 stimulation. Accordingly, loss of PI3K-C2γ does not affect insulin-dependent Akt1 activation as well as S6K and FoxO1-3 phosphorylation, but selectively reduces Akt2 activation, which specifically inhibits glycogen synthase activity. As a consequence, PI3K-C2γ-deficient mice display severely reduced liver accumulation of glycogen and develop hyperlipidemia, adiposity as well as insulin resistance with age or after consumption of a high-fat diet. Our data indicate PI3K-C2γ supports an isoenzyme-specific forking of insulin-mediated signal transduction to an endosomal pool of Akt2, required for glucose homeostasis.
Asunto(s)
Envejecimiento/genética , Glucógeno/metabolismo , Hepatocitos/metabolismo , Insulina/metabolismo , Hígado/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas de Unión al GTP rab5/metabolismo , Adiposidad/genética , Animales , Dieta Alta en Grasa , Endosomas/metabolismo , Factores de Transcripción Forkhead/metabolismo , Glucosa/metabolismo , Glucógeno Sintasa/metabolismo , Homeostasis , Hiperlipidemias/genética , Resistencia a la Insulina/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas Quinasas S6 Ribosómicas/metabolismo , Transducción de SeñalRESUMEN
Professional phagocytic cells ingest microbial intruders by engulfing them into phagosomes, which subsequently mature into microbicidal phagolysosomes. Phagosome maturation requires sequential fusion of the phagosome with early endosomes, late endosomes, and lysosomes. Although various phosphoinositides (PIPs) have been detected on phagosomes, it remained unclear which PIPs actually govern phagosome maturation. Here, we analyzed the involvement of PIPs in fusion of phagosomes with various endocytic compartments and identified phosphatidylinositol 4-phosphate [PI(4)P], phosphatidylinositol 3-phosphate [PI(3)P], and the lipid kinases that generate these PIPs, as mediators of phagosome-lysosome fusion. Phagosome-early endosome fusion required PI(3)P, yet did not depend on PI(4)P. Thus, PI(3)P regulates phagosome maturation at early and late stages, whereas PI(4)P is selectively required late in the pathway.
Asunto(s)
Lisosomas/metabolismo , Fagosomas/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , 1-Fosfatidilinositol 4-Quinasa/metabolismo , Animales , Línea Celular , Sistema Libre de Células/metabolismo , Cromatografía Líquida de Alta Presión , Endosomas/metabolismo , Immunoblotting , Membranas Intracelulares/metabolismo , Macrófagos/citología , Macrófagos/metabolismo , Espectrometría de Masas , Fusión de Membrana , Ratones , Microscopía Fluorescente , Microesferas , Fagocitosis , Fosfatidilinositol 3-Quinasas/metabolismoRESUMEN
Muscle wasting that occurs during aging or from disease pathology presents with an accumulation of lipid species termed ceroid or lipofuscin. This unique species of lipid has been characterized in various cell types but its properties and organization in skeletal muscle remains unclear. Using immunofluorescence and transmission electron microscopy, we were able to visualize and characterize an atypical lipid storing organelle in skeletal muscle. White myofibers contain two organelles at each pole of the myonuclei and red myofibers contain many of these structures in and around the perinuclear space. These organelles contain markers for late endosomes, are morphologically similar to multivesicular bodies, store lipid, and hypertrophy in aged muscle and a model of muscle wasting with an accumulation of large amounts of lipofuscin. Rapamycin treatment reduces the multivesicular body hypertrophy, restores late endosomal protein markers, and also increases the number and intensity of lipofuscin deposits. Together, these data demonstrate for the first time a perinuclear organelle in skeletal muscle that hypertrophies in muscle wasting phenotypes and is involved in endocytic lipid storage.
RESUMEN
Pompe disease is due to a deficiency in acid-α-glucosidase (GAA) and results in debilitating skeletal muscle wasting, characterized by the accumulation of glycogen and autophagic vesicles. Given the role of lysosomes as a platform for mTORC1 activation, we examined mTORC1 activity in models of Pompe disease. GAA-knockdown C2C12 myoblasts and GAA-deficient human skin fibroblasts of infantile Pompe patients were found to have decreased mTORC1 activation. Treatment with the cell-permeable leucine analog L-leucyl-L-leucine methyl ester restored mTORC1 activation. In vivo, Pompe mice also displayed reduced basal and leucine-stimulated mTORC1 activation in skeletal muscle, whereas treatment with a combination of insulin and leucine normalized mTORC1 activation. Chronic leucine feeding restored basal and leucine-stimulated mTORC1 activation, while partially protecting Pompe mice from developing kyphosis and the decline in muscle mass. Leucine-treated Pompe mice showed increased spontaneous activity and running capacity, with reduced muscle protein breakdown and glycogen accumulation. Together, these data demonstrate that GAA deficiency results in reduced mTORC1 activation that is partly responsible for the skeletal muscle wasting phenotype. Moreover, mTORC1 stimulation by dietary leucine supplementation prevented some of the detrimental skeletal muscle dysfunction that occurs in the Pompe disease mouse model.